Journal: The EMBO Journal

Article Title: Neuron‐specific translational control shift ensures proteostatic resilience during ER stress

doi: 10.15252/embj.2021110501

Figure Lengend Snippet: A Differentially expressed genes (DEGs) involved in oxidative stress‐related processes (Fig ), comparing TM‐treated Perk KO and WT neurons. Blue arrow: Ang. See also Appendix Fig . B, C Ang mRNA level in WT and Perk KO neurons and astrocytes ±20–24 h of TM‐induced ER stress, determined by mRNA‐seq ( N = 3). FPMK: Fragments per kilobase of transcript per Million. D, E Representative WB and quantification of ANG protein level ( N = 3). F, G tRNA unfolding in WT and Perk KO neurons ±20–24 h of TM‐induced ER stress ( N = 4). (F) Representative images obtained by high‐content microscopy of 1‐methyladenosine (m1A) immunofluorescence to determine tRNA unfolding. Dendrites (MAP2, white), nuclei ((Δ)Cre, red), unfolded tRNA (m1A, green). Scale bar: 25 μm. (G) Quantification of m1A intensity, normalised to untreated WT. H–M Abundance of small RNAs in WT and Perk KO astrocytes ( N = 3) and neurons ( N = 6) ± 20–24 h of TM‐induced ER stress, determined by high‐resolution automated electrophoresis. (H, K) Representative gel image. Electropherogram (I, L) and zoom (J, M) showing quantified fluorescence units (FU) of small RNAs, size ranges of tRNAs and tsncRNAs are indicated. Shown is the mean of the biological replicates. N–R Protein synthesis and ATF4 expression in WT and Perk KO neurons ±20–24 h of TM‐induced ER stress in the presence or absence of ANG‐I ( N = 3). (N) Representative images obtained by high‐content microscopy. Dendrites (MAP2, white), nuclei ((Δ)Cre, red) and de novo synthesis of proteins (puromycin, green). Scale bar: 25 μm. Quantification of the ER stress‐induced protein synthesis (O) and ATF4 response (Q). Data are normalised to WT without ANG‐i. Baseline level (without ER stress) is depicted by dashed line. Quantification showing the percentage change in the ER stress‐induced protein synthesis (P) and ATF4 response (R) induced by ANG‐i. (P) Calculated from data in (O): ANG − i + − ANG − i − / ANG − i − * 100 . (R) Calculated from data in (Q): ANG − i + − ANG − i − / ANG − i − * − 100 . Data information: Data are presented as mean ± SEM. N : Biological replicate. Relevant P ‐values are indicated: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Statistical analysis: two‐way ANOVA with Tukey's post‐hoc test (B, C, E, G, O, Q); nested t ‐test (P, R). Source data are available online for this figure.

Article Snippet: Primary antibodies used in this study for Western blot (WB) analysis or immunofluorescence (IF) were as follows: PERK (C33E10) (1:1000; Cell Signaling Technology; Cat# 3192, RRID:AB_2095847), pospho‐eIF2α (Ser51) (1:500; Cell Signaling Technology; Cat# 9721, RRID:AB_330951), eIF2α (1: 1000; Abcam; Cat# ab5369, RRID:AB_304838), puromycin (WB: 1:2500, IF: 1:500; Millipore; Cat#MABE343, RRID: AB_2566826), ATF4 (WB: 1:500, IF: 1:250; Cell Signalling Technology; Cat#11815, RRID: AB_2616025), GFAP (1:500; Sigma‐Aldrich; Cat# G3893, RRID:AB_477010), MAP2 (1:500; Abcam; Cat# ab5392, RRID:AB_2138153), ANG I (1:500; Santa Cruz Biotechnology; Cat# sc‐74,528, RRID:AB_2227157), 1‐methyladenosine (m1A) (1:500; MBL International; Cat# D345‐3, RRID:AB_2728758), GAPDH (1:2000; Millipore; Cat# MAB374, RRID:AB_2107445), NRF2 (1:250; Novus; Cat# NBP1‐32822, RRID:AB_10003994), HRI (1:750; Merck Millipore; Cat# 07–728, RRID:AB_441964), phospho‐mTOR (59.Ser 2448) (1:250; Santa Cruz Biotechnology; Cat# sc‐293,133, RRID:AB_2861149), mTOR (1:250; Santa Cruz Biotechnology; Cat# sc‐517,464), phospho‐p70 S6 Kinase (Thr389) (1:1000; Cell Signaling Technology; Cat# 9205, RRID:AB_330944), p70 S6 Kinase (1:1000; Cell Signaling Technology; Cat# 2708, RRID:AB_390722), phospho‐4E‐BP1 (Thr37 / Thr46) (1:1000; Cell Signaling Technology; Cat# 2855, RRID:AB_560835), 4E‐BP1 (53H11) (1:1000; Cell Signaling Technology; Cat# 9644, RRID:AB_2097841) and G3BP (1:100; BD Biosciences; Cat# 611126, RRID:AB_398437).

Techniques: Microscopy, Immunofluorescence, Electrophoresis, Fluorescence, Expressing

Journal: Cell Death Discovery

Article Title: N 1 -Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction

doi: 10.1038/s41420-023-01458-2

Figure Lengend Snippet: A Identification of m1A peaks in mouse cortical neuron mRNA. B Identification of the genes mapped to the m1A peaks. C OGD/R-induced peaks were divided into OGD/R-specific, Con-specific, and common peaks. The fold change of each peak was normalised and is denoted with the row z score. ‘OGD/R-1’, ‘OGD/R-2’, and ‘OGD/R-3’; Con-1’, ‘Con-2’, and ‘Con-3’ denote 3 biological replicates. D – F IGV displays the representative peaks of m1A-seq. D OGD/R-specific peak of Cwc25. E Con-specific peak of Btbd3. F Common peak of Traf3ip1. G GO analysis (top 10) and H KEGG analysis (top 10) of the m1A-modified genes on the cortical neuron transcripts. The results of the GO and KEGG analyses with enrichment scores and p values.

Article Snippet: Fragmented RNA was then incubated with the anti-m1A polyclonal antibody (MBL International, D345-3) in IPP buffer for two hours at 4 °C.

Techniques: Modification

Journal: Cell Death Discovery

Article Title: N 1 -Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction

doi: 10.1038/s41420-023-01458-2

Figure Lengend Snippet: A Overall abundance of m1A levels in cortical neuron transcripts and after OGD/R induction. The box limits represent the upper quartile, the median, and the lower quartile. The extremes represent the maximum and minimum values ( p < 2.2e-16, Kruskal–Wallis test). B Representative analysis of the modified region of the m1A transcript. C Transcripts are m1A modified during OGD/R induction. D The proportion of transcripts carrying 1, 2, 3, 4, 5, and 6 or more peaks in neurons and after OGD/R induction. E The distribution and F density of the m1A peaks on each chromosome. G GC content in the m1A sequence and non-m1A sequence during OGD/R induction. G + C% represents the percentage of GC dinucleotides in the sequence. H qRT-PCR showed changes in the expression of the methylation-related enzymes Trmt6, Trmt61a, Trmt10c, Alkbh3, and Ythdf3 in mouse cortical neurons after OGD/R induction. I , J Western blot showed changes in the expression of the methylation-related enzymes Trmt6, Trmt10c, Alkbh3, and Ythdf3 in mouse cortical neurons after OGD/R induction. Data analysis was performed using Student’s t test. The data are presented as the means ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. n.s. no significant difference.

Article Snippet: Fragmented RNA was then incubated with the anti-m1A polyclonal antibody (MBL International, D345-3) in IPP buffer for two hours at 4 °C.

Techniques: Modification, Sequencing, Quantitative RT-PCR, Expressing, Methylation, Western Blot

Journal: Cell Death Discovery

Article Title: N 1 -Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction

doi: 10.1038/s41420-023-01458-2

Figure Lengend Snippet: A Identification of differentially modified m1A peaks (red: upregulated, green: downregulated) (fold change ≥ 2, p ≤ 0.00001). B GO analysis of differentially modified upregulated m1A peak genes and C downregulated m1A peaks. D KEGG pathway analysis of differentially modified upregulated m1A peaks and E downregulated m1A peaks. F PPI network analysis of the differentially modified m1A peaks and G core genes of the top 20 interactions. The relationship of methylation regulation is colour coded. Red: upregulated genes. Blue: downregulated genes. H , I Degree of m1A methylation of the downregulated peak Nrgn during OGD/R induction. J , K The degree of m1A methylation of the Tead2 upregulated peak during OGD/R induction. GO and KEGG analyses with enrichment scores and p values. MeRIP-RT-PCR data were analysed using Student’s t tests. The data are presented as the means ± SD, n = 3. ** p < 0.01, **** p < 0.0001.

Article Snippet: Fragmented RNA was then incubated with the anti-m1A polyclonal antibody (MBL International, D345-3) in IPP buffer for two hours at 4 °C.

Techniques: Modification, Methylation, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death Discovery

Article Title: N 1 -Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction

doi: 10.1038/s41420-023-01458-2

Figure Lengend Snippet: A Position distribution of m1A peaks in the transcript. mRNA is divided into 5 regions: 5′ UTR, start codon, CDS, stop codon, and 3′ UTR. According to previous studies, the regions 100-nt upstream or downstream of the start and stop codons are defined as the start codon and stop codon. B Percentage of m1A peaks distributed in different positions of the transcript. The data are presented as the means ± SD, n = 3. C The higher the gene expression, the higher the proportion of methylation. RNA-seq expression levels were used to separate genes into deciles, and the methylation rate of RNA-seq genes in each decile was assessed. D Relationship between m1A modification and gene expression. According to the m1A-seq data, each transcript was defined by whether there was a methylation-modified gene, and the expression of each gene was used to draw a box plot ( p < 2.2e-16 and p < 1.2e-09, respectively; Kruskal–Wallis test). E m1A modifications in different regions of the transcript have different effects on gene expression. According to the area where the peaks were located and the RNA-seq expression data, a box plot was drawn ( p = 3e-06, p < 1.2e-09, respectively; Student’s t test). F Effect of the number of peaks on gene expression. Classification was based on the number of peaks in the transcript and the RNA-seq expression data, which were used to draw a box plot. The box limits represent the upper quartile, the median and the lower quartile. The extremes represent the maximum and minimum values. m1A: transcripts with m1A modification; non-m1A: transcripts without m1A modification. n.s. no significant difference.

Article Snippet: Fragmented RNA was then incubated with the anti-m1A polyclonal antibody (MBL International, D345-3) in IPP buffer for two hours at 4 °C.

Techniques: Gene Expression, Methylation, RNA Sequencing, Expressing, Modification

Journal: Cell Death Discovery

Article Title: N 1 -Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction

doi: 10.1038/s41420-023-01458-2

Figure Lengend Snippet: A The differential distribution of m1A peaks correlates positively with the gene expression. A scatter plot was drawn based on the height of the methylation peaks and the level of gene expression. cor.test was applied using the Pearson method (Pearson r = 0.054, p < 0.0001). B A related heat map of methylation and expression was generated for the 10 genes that exhibited a significant change in both the m1A level and mRNA transcript abundance after OGD/R induction and in primary mouse cortical neurons. C MeRIP-RT-PCR shows the changes in specific m1A modification levels during OGD/R induction. D qRT-PCR shows the changes in specific gene expression during OGD/R induction. E Changes in the expression of 5 specific genes in 10 control samples and 12 disease samples from the GSE19587 database. F Changes in the expression of 4 methylation modification regulatory enzymes in 4 control samples and 4 disease samples from the GSE19587 database. ns no significant difference. Hyper-up: highly methylated and highly expressed gene. Hypo-down: Weakly methylated and weakly expressed gene. The data are presented as the means ± SD, n = 3. The p value was calculated using Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.0001.

Article Snippet: Fragmented RNA was then incubated with the anti-m1A polyclonal antibody (MBL International, D345-3) in IPP buffer for two hours at 4 °C.

Techniques: Gene Expression, Methylation, Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Modification, Quantitative RT-PCR, Control

Journal: Cell Death Discovery

Article Title: N 1 -Methyladenosine modification of mRNA regulates neuronal gene expression and oxygen glucose deprivation/reoxygenation induction

doi: 10.1038/s41420-023-01458-2

Figure Lengend Snippet: Ten genes exhibiting a significant change in both m1A levels and mRNA transcript abundance in OGD/R induction and mouse primary cortical neurons.

Article Snippet: Fragmented RNA was then incubated with the anti-m1A polyclonal antibody (MBL International, D345-3) in IPP buffer for two hours at 4 °C.

Techniques:

Journal: STAR Protocols

Article Title: Protocol to analyze and quantify protein-methylated RNA interactions in mammalian cells with a combination of RNA immunoprecipitation and nucleoside mass spectrometry

doi: 10.1016/j.xpro.2022.101268

Figure Lengend Snippet:

Article Snippet: 1-methyladenosine ribonucleoside , Cayman Chemical Company , 16937.

Techniques: Recombinant, Protease Inhibitor, Pore Size, Targeted Proteomics, Software

Journal: Journal of medicinal chemistry

Article Title: 2-Triazole-Substituted Adenosines: A New Class of Selective A 3 Adenosine Receptor Agonists, Partial Agonists, and Antagonists

doi: 10.1021/jm0608208

Figure Lengend Snippet: (A) Docking complexes of compound 10, 2-(4-cyclopentylmethyl-1,2,3-triazol-1-yl)-N6-methyladenosine. (B) Superimposition of Cl-IB-MECA in red and compound 10 in color by atom type. Residues that were within 5 Å to the ligand in this putative binding site were L91 (3.33), T94 (3.36), H95 (3.37), Q167 (EL2), F168 (EL2), M172 (EL2), S181 (5.42), M177 (5.38), V178 (5.39), F182 (5.43), W243 (6.48), L246 (6.51), S247 (6.52), N250 (6.55), C251 (6.56), I268 (7.39), S271 (7.42), and H272 (7.43). The ligand is represented by a ball-and-stick model. The H-bonds are indicated with yellow dots. By use of the MOLCAD ribbon surface program, the A3AR is shown in a ribbon model with different colors for each TM (TM1, red; TM2, orange; TM3, yellow; TM4, green; TM5, cyan; TM6, blue; TM7, purple; H8, violet).

Article Snippet: Compound 10 , 2-(4-cyclopentylmethyl-1,2,3-triazole)- N 6 -methyladenosine, was constructed with the use of the Sketch Molecule of SYBYL 7.1 (Tripos Inc., 1699 South Hanley Road, St. Louis, MO 63144).

Techniques: Binding Assay